Antiricketic substances and the production thereof



lPatented Aug. 14, 1928.

UNITED STATES 1,681,120 PATENT AUGUST J". PAGINI, OF CHICAGO, ILLINOIS, ASSIGNOH TO CHARLES M. RICHTER, OF

CHICAGO,

ILLINOIS.

AN'IIRIGKETIC SUBSTANCES AND THE PRODUCTION THEREOF.

No Drawing.

This invention relates to anti-ricketic prod nets and the like, and with regard to certain more specific features, to the production of anti-ricketic and similar substances.

5 Among the several objects of the invention may be noted the treatment of substances which elaborate g'rowth'producing sub stances, hereinafter for brevity termed Vita min a, to produce anti-ricketic substances which include the so-called vitamin D, without the use of radiation of wavelength shorter than about 3022 Angstrom units.

Another object of the invention is to pro vide means of the class described, by which conunercial production ofanti-ricketic food and medicinal products may be practically accon'iplished on a substantial scale of operations.

Another object of the invention is the pr0- vision of improved means for making concentrated water-soluble anti-ricketic medicinal and other products, whichinclude said vitamin D, from merely fat-soluble substances of a corresponding nature.

Another object of the invention is the production of water-soluble fractions adapted to be useiil hypodermically as anti-ricketic injections.

Other objects will be in part obvious an in part pointed out hereinafter.

The invention accordingly comprises the elements and combinations of elements, steps and sequence of steps, features of construction and arrangements which will be exemplilied in the articles hereinafter described,

and the scope of the application of which will be indicated in the following claims.

Qlt can be shown that certain growing, living things are apparently sources of a g ;rowt]i-producing substance, apparently related to but not identical with vitamin A, and hereinafter for brevity termed vitamin a. The more rapidly growing things, in general, appear to be the best sources of this growth producing substance, as for example, typhoid bacilli, rapidly growing sarcomata, certain of the fungi, certain of the ascomycetes and otl; The various vitamins, under suitable producing properties possessed by the classes Application filed August 24, 1927.

3 methods of animal experimentation, in which Serial No. 215,264.

of vitamins enumerated, the separate groups show other characteristics by which their distinction becomes possible. Thus, vitamin A is growth-producing, is also anti-xerophthalmie and is considered fat soluble. Vitamin B is growth-producing, anti-heriberic, and water soluble. Vitamin D is growth -producing, anti-ricketic and fat soluble. The classes of materials serving as sources for vitamin c as the term is used in the present application are lrnown to furnish water soluble vitamin B, but the presence of vitamin A, which is probably complex and consists of at least both a growth-producing and an anti-xeroplithahnic fraction, has heretofore been considered absent, or present in scarcely appreciable amounts. The matter of suitable extraction of the fat soluble vitamin A is acknowledged to be diflicult, certain organic solvents being effective when used with one material and ineffective When used with another. This same ditliculty is experienced with the substances designated vitamin a. Novel means for accomplishing extractions are given hereinafter.

For example, one of, the fungi, agaricus oampestris (ordinary ineadowngaric), may be used as a source of growth-producing mate rial (vitamin c). For this purpose it is finely minced in its fresh state and extracted with a mixture of acetone and alcohol in the following proportions:

Ewample 1.

Agaricus eampestris (fresh) two kilograms Extractant: alcohol (ethyl,

absolute) -500 cc. acetone, Kahlbaum (free from methyl alcohol) 1000 cc.

The extraction is made by reflux at reduced pressure in a period of six hours. At the conclusion of the extraction, the solvent is decanted from the residue and is evaporated at reduced pressure in vacuum, whereby a fatty residue is obtained weighing of the order of 0.7833 granui. This residue is dissolved in alcohol (absolute ethyl) in proportions adapted to make a solution, each cubic centimeter of which contains ten milligrams of residue. This alcoholic solution is then treated by shaking with fullers earth in the ratio of one gram fullers earth per cubic centimeter of solution and the alcohol per-- mitted to evaporate, thus distributing ten milligrams of ag'aricus extract throughout one gram of fullers earth.

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The growth-producing, or inducing substance appears now to be transfixed to the fullers earth, presumably by adhesion, thus affording a comparatively quantitative measure of effectiveness. The earth when fed to rats which have been placed on a growthfree diet,owing to lack of vitamin A, will cause increase in growth, thus demonstrating that a growth-producing substance is present in the extract, this substance belonging presumably to the group of vitamins known as vitamin A. If the extraction is carried on with benzene instead of acetone and alcohol mixture, a larger amount of fatty residue is obtained, weighing of the order of 1.5639 grams. Comparison of the residue obtained by the acetone-alcohol extraction with the residue obtained by the benzene extraction, bothresidues being fed to experimental rats kept on a diet adequate in every respect save for the absence of vitamin A, discloses considerable anti-xerophthalmic effect in the case of the benzene residue and a negligible anti-xerophthalmic effect in the ease of the acetone-alcohol residue, growth-producing properties being present in both instances. It thus appears that vitamin A may be composed of several growth-producing components, the anti-xerophthalmic effect belonging to one of these components, or comprising possibly a separate vitamin. As stated above, for the purpose of brevity, the growthproducing fat soluble and apparently antixerophthalmic lacking substance is identified by the special designation vitamin a.

The growing things mentioned above and others will produce similar extracts but they are not all equally potent, as the following table shows:

Relative growth Substance d ti n ower 1. Typhoid bacilli 2. Brown rot ofpeachea.

3.Sarcomatous tissue.

4.Agarieus eampestris 5. Ergot ofrye As an example of the mode of forming another extract, the following short method is given:

v Example 2.

tissue, agaricus campestris, ergot of rye, and undoubtedly to be obtained also from sources belonging to the classes of which the substances named are but class examples, have been found to be devoid of anti-ricketic prop erty, and therefore, devoid of vitamins of the D group. One of the objects of this invention is to endow the growth-producing quality of substances of the class above set forth, with an antimicketic property.

To accomplish the above end, the water insoluble residues containing vitamin a, of the class above set forth, are treated with certain light radiatimi, excluding therapcutically active ultraviolet radiation. By ultraviolet radiation is meant that radiation which is outside the visible spectrum at its violet end and starts at about i000 Angstrom units to the region of the X-ray at about 500 .iingstrom units. Converted to units of wave length, this amounts to wave lengths from 0.41 of :1 micron to 0.05 of :1 micron of 0000+ of a centimeter to .000005 of a centimeter. It is to be understood that l intend to use irradiation including wave lengths below and/ or beyond the ultraviolet, that is, longer and/or shorter wave lengths. The salient point is that my method exchidcs ultraviolet.

Biologically useful ultraviolet radiation is differently designated, the basis for the dcsigp nation residing in the fact that ordinary window gass considered opaque to the c llective ultraviolet radiations. .liy c'll'ective ultraviolet radiation is meant here that region which is curative for rickets in animals and in humans, and which has been demonstrated to include wave-lengths beginning at. about 3022 Angstrom units and extending possibtv as far as 2000 llngstrom units (0.302 to 0.2 microns, or 0.0000302 to 0.00002 centimeters). Ordinary incandescent illuminating sources. such as tungsten filament lamps commonly called lilazda lamps, emit ultraviolet radiation; but inasmuch as these rays must pass through the glass bulb in which the filament is contained. the shorter and biologically effective radiations are obstructed. Such sources of radiation emitting small. subintensive amounts of glass filtered ultraviolet are not considered sources of biologically or therapeutically active ultraviolet radial ion in the art. Ultraviolet radiation as the phrase is ordinarily used means radiation of wavelength shorter than that filtered b ordinhrv glass and such as is obtained from a mercury vapor lamp in quartz. ordinary glass having the effect of removing the nccessa ry radiation.

hen the extracted material is obtained. in the manner specified. from substances for which class examples have been set forth. l have discovered that the exposure of such material to certain radiations exclusive of the range which by common consent is dclinod as designating the biologically and therapeutically active ultraviolet radiation, occasions in the material an apparent conversion of fat soluble vitamin e to fat soluble vitamin D, the growth-preducing properties of the vitamin a are lost and become replaced by pronounced anti-ricketic vitamin D properties.

For example, the organic solvent extract from agaricus campestris loses its vitamin c properties and acquires instead anti-ricketic vitamin 1) properties when exposed to light as follows:

Example 3.

The organic solvent extract containing vitamin aiis placed in a covered glass Petri dish, and is exposed 210 or more minutes at 20 inches from a source 01 radiation including Wavelengths down to about 4:000 Angstrom units. It is not necessary that the receptacle containing the material to be treated be glass covered, any open receptacle serving just as Well. The use of a glass covered receptacle simply establishes the effectiveness at radiations other than those in the region heretofore considered as the sole biologically active ultraviolet region. For example, the intensity of the. light used at the source was that obtained from a 1500 watt tungsten lilament incandescent daylight lamp. To be of maximum effectiveness the radiation must be intensified in the limits of the visible spec trum and must be devoid of ultraviolet. Passing the radiation from a 1500 watt til? sten filament incandescent day-light lamp, through a lilter opaque to the ultraviolet accontiplishes the result. The lilter used may be a solution of cupric chloride, live grams of the anhydrous salt dissolved in one liter oi? water, and contained ina glass trough in a layer approximately three centimeters in thickness. Other methods oi filtration may be used, as for example Water, or other solutions of different organic, or inorganic dyes and substances, such as picric acid dissolved in water, potassium chromate in Water, or cobalt chloride in acetone. Another method of filtration consists in the use of specially prepared glasses, colored, or otherwise made opaque to ultraviolet radiation. Sources of light other than tungsten filament lamps also produce the results, as for example Cooper Hewitt lamps in glass, carbon and other are lamps. Another method ol furnishing adequate radiation consists in the use of relatively monochron'iatic light sources, such for example as a. neon or other gas lamp made incandescent by high frequency currents or other means. Any source of light may be used, even though it is inherently rich in the ultraviolet, if the ultraviolet is screened out before the rays reach the substance being treated. The pri mary point is that growtli-producing substances rich in vitamin a develop anti-ricketic property, or become rich in. vitamin D by means of rays more desirable than ultraviolet.

It is to be understood that ordinary sun light will accomplish the same end, but it is slow in action and inconstant in its intensity. Hence artificial lighting methods are preferable. It is to be understood that artificial lighting methods are to include such as cause a concentration of sunlight beyond its ordinary powers, whereby its eiiicacious action is increased for the purpose in hand. Examples would include concentrating, retracting lenses and reflecting mirrors. However, in all such cases, as stated above, the ultraviolet rays are to be screened out,

The action of the rays can be interpreted in terms of photo--chemistry and probably in volves electronic Ire-orientation in the atoms of the molecules undergoing activation. By activation is meant, for example, the apparent replacement of at least some oi vitamin 0; with vitamin l), or the production of vitamin D without the pro-existence oit vitan'iin a. I have discovered that energy sources other than ultraviolet radiation of the biologically ei'tcctive range are capable oil? producing the activation referred to, such sources including radiation from 3022 to 30,000, ilugstroms units which includes the infra-red Wavelengths.

If, as has been siiiggested in the literature referring to vitamins, a unit of vitamin is accepted as representing that amount which when fed daily, just sullices to support a gain in weight of three grams per week in an experii'nental animal (white rats of known pedigree, age 25 to 28 days) properly prepared ilor such testing, the vitan'iin a content ot' some of the substances examined appears to be approximately as "follows, ex:- pressed in units of vitamin per gram oil extract of the material Approximate unit Extract fi'oiiiof vitamin Vitamin D 11 pt am of extract 1. Brown rot oi peaches 6, 000 2. Agni-ions cmnncstris 10, 000 3. Ergot of rye .1 30,001)

When subjected to the process by the 1. Brown rot of peaches .i 10, 000 2. Agarieus campcstris 20,000 3 Ergot of rye 60,000

The irradiation produc s a change in \vh ich the amount of vitamin a lost becomes replaced by a proportionate amount of vitamin it) gained. This is possibly due to a conversion or transmutation of vitamin a into vitamin D.

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Excessive exposures, 400 hours amounting to more than ten times that necessary to produce the vitamin D el'l ect, do not impair the production of vitamin D.

lhe photosynthesis of anti-ricketic vitamin can be accomplished by exposiny' suitable material to radiation mentioned whether or not these substances contain vitamin a. Thus for example, oleomargarine, a food completely devoid of vitamin A, and devoid of vitamin (4, becomes anti-ricketic when subjected to theprocess as illustrated in Example 3 in which the radiation used is infra-red rays, and various grain products such as oats, containing vitamin A and vitamin a become antia'iclietic by the same process; in the case of these products, however, without the loss of their anti-xerophthalmic power, where such power already exists.

l he reason for treating the growth-producing and like substances with light oil wave lengths which exclude the ultraviolet is that a greater amount oi? material may be treated at one time without the use of impractical and expensive equipment, such as is required when treating with ultraviolet rays. Heretotore treatments have been made on the theory that the ultraviolet ray is the only one which will accomplish the end, but I have discovered that the other rays specified have the desired efiiect and that the same end can be accomplished more expeditiously therewith. For instance, some of the known ways of producing ultraviolet rays are by means of the quartz mercury vapor lamps, which are expensive and inconvenient devices to operate where commercial operations are to be carried on.

Another point with regard to treatment of substances by ultraviolet rays is to be noted. If food or the like is exposed to these rays for purposes corresponding to the above,

especially after inadvertent over-exposure has been made, it has been found that an undesirable bleaching result is had, as well as an undesirable effect which renders the food less palatable and in many instances causes a. definite distaste'tulncss. Oils, for example, acquire a decided acridity of taste and smell, making them less pleasingly palatable. By treating the residue or foods impregnated with said residue, or the other classes of substances mentioned with rays other than ultraviolet, the improved desirable anti-ricketic conversion is accomplished, without the undesirable. properties attending the use of ultraviolet rays.

Another disadvantage in using ultraviolet rays, not had by using the longer of the other rays prescribed herein, is that operators are subjected to conjunctivitis. By using said longer rays, operators are subjected to no inconvenience or ill effects.

Another object of this invention, per se, and in combination with the above is to convert The irradiated water-insoluble or fatty residue is treated with a. water solution oi. sodium taurocholate and sodium lycocholate, of the 'lollowing 'iroportions:

Parts. Sodium taiu'ocholate 1 Sodium glycocholate 3 Water ii A satistactory solution is e'llected when the fatty residue treated with approximatcly 20 times its weight of solution; thus, for 0.5 gam of fatty residue, 10 cubic centimeters of the solution sufiice. The treatn'ient is a digestion, effected in a sealed glass tube immersed in boiling water for at least 8 hours. A dark brown solution, slightly turbid and containing' a white sediment results. At the conclusion of the digestion, the solution is filtered and evaporated to dryness. The residue is dissolved in grain alcohol, gentle heating effecting;- complete solution. illeutral lead acetate is added to the alcoholic solution, which precipitates lead glycocholate. r'iilter removing the lead glycocholate by filtration, basic lead acetate and ammonia are added to the remaining;- alcoholic solution, which precipitates the taurocholate. This is removed by filtration. All excess of lead is removed from the alcohol solution by passing hydrogen sulphide through the solution which is first made -faintly acid to litmus with hydrochloric acid. Lead separates out as lead sulphide and is .tiltered oil. The remaining alcoholic solution is evaporated to dryness, and the residue, which is water soluble, found to be strongly anti-ricketic. For further purification, the residue in water may be dialyzed against water. it is to be understood that other substanccs may be used to accomplish the conversion, ha ving the properties of sodium taurocholate and sodium glycocholate, such as other dcrivaties of cholic and similar acids.

It should be understood that the method of converting water-insoluble substances to water-soluble substances can be used independently oi the method of converting a growth-producing or other substance i nto an antiricl etie substance by said means including the use of non-ultraviolet rays. For instance, extracts oi cod liver oil rich in llnl vitamins A and D, may be rendered Watersolubleby the process above described, with out light treatment, and therefore without h aving changed their vitamin A. content to a vitamin D content and Without deleteriously affecting); said vitamin D centcnt. Examples could be multiplied.

The Water-soluble fractions, obtained by the methods above set out for making an antiricketic medicinal product, instead of being evaporated, may be used hypoderi'i'iically as an antiicketie injection, completely capable of affording the anti-ricketic benefits herein (1 scribed. The injections may be made in amounts up to 2000 milligrams Without deleterious effects.

Having described the details of the method for efiecting all ofthe above, the following is to be made clear:

Foods are rendered anti-ricketic, either by applying the growthproducing or like substance or substances to the food by Wet or dry mixing and then exposing the thus treated food to the non-ultraviolet rays in order to render said substances anti-ricketic, or the substances may be first rendered anti-ricketic by exposure to said rays and then applied to the food by wet or dry mixing or, the food may be exposed directly to the non-ultr'.- violet 'ays, as in the case of oleomargarine. Mixing may be accomplished mechanically or otherwise.

Taking for example oats, they are impregnated with the growth-producing substance (in extract or other form) and then exposed to the non-ultraviolet rays by continuous passage on belts orthe like under suitable lamps. Or, they are impregnated with a sub stance which has been previously rendered anti-ricketic by exposure to said rays or, they are exposed directly to the non-ultraviolet rays.

The same variations of method hold with respect to the manufacture of concentrated medicinal products, although the direct methods, hereinbeforedescribed, are preferable in the case of the manufacture of said medicinal products.

In this application, the term vitamin D concentrate refers to a substance having a vitamin D activity of a hi gher order of magnitude than is found in nature.

In view of the foregoing, it Will be seen that the several objects of the invention are achieved and other advantageous results at tained.

As many changes could be made in carrying out the above preparations withoutdeparting from the scope of the invention, it is intended that all matter contained in the above description shall be interpreted as illustrative and not in a limiting sense. I claim: l l l. The method. of preparing materials con" products, said method comprising the artificial application of infra-red rays to said materials.

4. The method of preparing materials containing vitamins a to exchange at least some of said vitamins or for vitamins D, comprising applying rays longer than about 3022 Angstriim units to said materials.

5. The method of preparing materials con taining vitamins a to exchange at least some of said vitamins a for vitamins D, comprising applying rays to said materials having Wave lengths of the order of 3022 Angstrom units to about 30,000 Angstrom units.

6. The product including a vitamin D concentrate, said concentrate being in Water-soluble form.

7. The method of manufacturing an antiricketic food comprising extracting a growthproducing substance, treating the growthproducing extracted substance until at least a partial replacement by an anti-ricketic sub stance is obtained and applying the antiricketic substance to a food.

8. The method of manufacturing an antiricketic food comprising extracting a growthproducing substance, applying said substance to a food, and treatmg the growth-producing substance until at least some of it becomes replaced by an anti-ricketic substance, said;

substance, said treatment being accomplished by exposure to infra-red rays. i

10. The medicinal product comprising a vitamin D concentrate, said concentrate being in Water-soluble amorphous powder form.

"ll. The medicinal product comprising; a

vitamin D concentrate in a tablet, said concentrate comprising a water-soluble amorphous powder.

12. The method of manufacturing a medicinal anti-ricketic product comprising extracting a growth-producing substance, treating the growth-producing substance until at least some of it becomes replaced by an antiriclietic product after extraction, saidtreatment being accomplished by exposure to rays longer than about 3022 fingstrom units.

13. The method of manufacturing a medicinal anti-ricketic product comprising e;; t-racting a growth-producing substance, treating the growth-produing substance until at least some ot'it becomes replaced by an antiricketic product Zt'litGl' extraction, said treatment being accomplished exposure to rays longer than about'3022 Angstrom units and fractinating the activated product for use hypodermically.

14:. The. method of manufacturing a me dicinal anti-ricltetic product comprising the preparation of a growtlrproducing substance, treating the growth-producing. substance to produce an anti-ricketic product,

said treatn' ent being accomplished by exposure to rays longer than about 3022 ingstrem units, and: reducing, atleast part of the activated product to a water-solublesubstance by the use of a chol'ate derivative.

15. The method of manufacturing a medicinalTanti-ricketic product comprising the preparation of a growth-producing sub stance, treating the growth producing sub stance until it becomes replaced by an antirlcltetic product, said treatment being accomplished by exposure to rays longer than 3022 Q n u n-ngstrom rants, and reducing at least part of the activatedproduct to a water-soluble substance by the use of a cholate derivative and reducing the same to a powder.

16. The methodof manufacturing an antiricketic product comprising extracting a growtlrproducing material by means of a solvent, evaporating the solvent, whereby a residue is caused to remain, and treating the residue with a radiation of wave-length longer than about 3022 Angstrom units, whereby at least some ofthe growth-producing substance is replaced by an anti-ricketic product,

17. The method of manufacturing a watersoluble anti-ricketic product comprising extracting a growth-producing material by means otl a solvent, evaporating the solvent, whereby a fatty residue is caused toli main, treating the residue with a rad-iation of wavelength longer than about 3022 ingstroin units, whereby at least part of the growth producing substance becomes anti-ricketic,

and treating said fatty residue with a cholate derivative in order to render it water-soluble, whereby said anti-ricketic substance is made more assimilable.

18. The method of manufacturing a watersoluble anti-ricketic product comprising extracting a growth-prodiicing material by means of a solvent, evaporating the solvent, whereby a 'liatty residue is caused to remain, treating the residue with a radiation ol wavelength longer than about 3022 Angstrom units, whereby at least part of the growth producing substance becomes anti-richetic, and treating said tl atty residue with a cholate derivative, whereby said anti-ricketic substance is made more assimilable.

19. The method of manufacturing a water soluble anti-ricketic product comprising extracting a growth-prodiicing material, thereby producing a residue, treating the residue with radiation ol. wave-length. longer than about 3022 dngstrtim units, and treating the residue with a cholate derivative.

20. The method of treating materials containing vitamins a, to replace at least some oi said vitamins with vitamins D, comprising the artificial application of rays longer than about 3022 rlingstrtim units tosaid materials, whereby. said materials are caused to include vitamins D.

21. The method of treating materials containing an anti-xerophthaln1ic component and i an antixerophthaln'iic lacking but growth producing component so that at least part of said anti xerophthalmic lacking and growth producing component becomes replaced by an anti-ricketic component withoul deleteriously all ecting said antirxerophthalmic co 'nponent, comprising concentrating rays on said materials longer than about 3022 Angstrom units.

22. The method oftrcating. n'iaterials con taining an.anti-Xerophthalmic component and an antixerophthalinic lacking but growth producing con'iponent so that at least part of said anti-Xerophthalmic lacking and growth producing con ponent becomes replaced by an anti-nicketic component without deleteriouslyattecting said anti-xerophthalmie coin ponent, comprising. conceiurating infra-red rays on said materials, extracting one of the materials.bymeans of a solvent, evaporating thesolvent, whereby a fatty residue is caused to remain, concentrating infra red rays on said residue, and treating the fatty residue with acholate derivative to render it a watersoluble substance, whereby said anti-ricketic ltll) 

